With the increasing growth of population, declining arable land, and tightening of other agriculture-related resources, supply of crop food in the whole world is facing a huge challenge. Therefore, it becomes highly needed to increase and sustain crop production and improve crop quality. However, a variety of environmental stresses, such as drought, flood, freezing, heat, and pathogens, have greatly impacted yield and quality of crops.
Plant genetic engineering utilizes recombinant DNA technologies to modify and recombine genes in order to confer new traits to transgenic plants and obtain new varieties with higher yields, improved quality and stress tolerance. R&D on plant genetic engineering has advanced rapidly ever since the transgenic tobacco and potato were obtained in 1983. As of 1997, a total of about 25,000 field trials on genetically modified (GM) crops have been carried out globally, which involved 10 countries, 60 crops and 10 traits (Cheng et al., 2014). To date, a large number of high-yield, high-quality new varieties of crops with herbicide tolerance, disease resistance, insect resistance, and stress tolerance have been bred and broadly applied in agricultural production (Hou et al., 2005).
A large number of genes with high potentials have been identified in recent years, which include Arabidopsis stress-induced transcription factors DREB1A (Kasuga et al., 1999) and CBFs (Hsieh et al., 2002a; Hsieh et al., 2002b), etc. Constitutive overexpression of these genes in transgenic plants could result in improved phenotypes such as better stress tolerance and delayed plant senescence, which are frequently accompanied, however, by unfavorable effects on the growth and development under normal growth conditions, such as shorter height, retarded growth, or reduced yield. It is expected that the practical values of these transgenes could get significantly boosted if they are up-regulated specifically at certain developmental stage or under stress conditions while remaining down-regulated under normal conditions. As such, tissue-specific promoters are conventionally employed to drive expression of target genes (Kasuga et al., 1999; Lee et al., 2003; Kovalchuk et al., 2013; Wang et al., 2013). However, this approach has limitations. Firstly, only a limited number of tissue-specific promoters have been identified and applied to date, which generally have low promoter activity and low tissue specificity. Low promoter activity may result in the expression of target genes below the level required for functioning. Secondly, transgenes with breeding potentials need to be transcribed and translated into proteins in order to regulate the growth and development of transgenic plants after their integration into the genomes, and it is common for most genes that both the post-transcriptional and post-translational regulations play important roles in modulating gene expression and functionalities. Thus the only use of specific promoters is far from ideal for this purpose, but this weakness can be compensated by protein-level regulation, which thus has great potentials.
Recently Jang et al. reported that ubiquitin-like domain 1 (UBA1) and 2 (UBA2) of the Arabidopsis RAD23a protein could serve as stabilizing signals to significantly increase the stability of HFR1 and PIF3, two short-lived transcription factors, leading to a more pronounced phenotype of the transgenic plants than that of the plants merely overexpressing these transgenes. The UBA domain of the Arabidopsis DDI1 protein could also extend the half-life of the short-lived protein JAZ0.1 that is implicated in the jasmonate signaling pathway. It was proposed that fusion of UBA domain with other short-lived proteins to increase their stability may benefit the fundamental research of these proteins and may be used for modification and improvement of crop traits (Jang et al., 2012). Thus fusion of a polypeptide sequence that specifically regulates gene expression on protein level with other target proteins may result in accumulation of the target proteins under certain developmental stages or under specific stress conditions while still remaining low expression under other conditions. It helps to conditionally control the expression of genes with breeding potentials, resulting in a better control of the development of transgenic plants and leading to increased yields and improved qualities. To date there have been only limited cases where such polypeptide sequences are identified and applied in real agricultural practices, yet it is expected that this approach will play a significant role in the improvement of crop traits and the breeding of new varieties.
Delay of senescence is closely associated with the increase of crop yield and quality. In food crops and major cash crops, the assimilation products in functional leaves and the accumulated nutrients in senescent leaves are continuously allocated to yield organs during their development. Premature senescence thus has a disadvantageous impact on crop yield and quality. For instance, premature leaf senescence and hypofunction during the late development stage of some hybrid rice varieties leads to low seed-setting rates, which severely limits the further realization of yield potentiality (Gan et al. 1997). Furthermore, leaf senescence in green-leaf crops not only has negative impact on overall yield and quality but also directly affects other factors such as harvest yield, postharvest quality, and shelf life. It was reported that the postharvest loss of vegetables in China reached 10% -50% (Zhang et al. 2009). Additionally, for flowering plants, the senescence of leaves and floral organs directly affects their ornamental values and selling prices. Thus delay of senescence can potentially affect the yield and quality of almost all major crops and commercial plants.
Senescence is the final stage of leaf development, and as an active physiological process, it involves increased expression of a large number of senescence-associated genes (SAGs), which are implicated in the control and regulation of the senescence process. During senescence, the metabolism process inside the plant cell is extremely active and a large amount of assimilation products accumulated in leaves, including degradable cellular components, are degraded and transformed in an efficient and ordered manner, and then transported to the newly formed organs, especially the seeds and fruits (Buchanan-Wollaston et al., 2005; Lim et al., 2007; Lin et al., 2004). Leaf senescence is cooperatively regulated by internal signals (leaf age-correlated signals and levels of endogenous hormones, etc.) and various external factors (temperature, light, stress, etc.). Endogenous hormones are one of the major factors influencing leaf senescence, and regulation of leaf senescence is closely correlated with cytokinins. Tang et al. (1998) found that exogenous application of phenylurea cytokinin (4-PU-30) had significant green-sustaining and senescence-delaying effects on the leaves of hybrid rice, and additionally improved the photosynthetic rates, facilitated grain-filling and dry matter accumulation, which ultimately led to an increase of grain weight and yield. Liu et al. (2001) reported that spraying of lawn grasses such as heaven grass and Manila grass with different concentrations of GA3, BA or PP333 all resulted in delay of senescence, prolonging of green period, and enhancement of cold tolerance. Leaf senescence is also a highly programmed process affected by external factors. Unfavorable conditions such as pathogen infection, shade, heat, drought and flood may all lead to premature senescence of the plants. It has been showed that during rice grain-filling, high temperature could cause accelerated senescence of rice leaves, which in turn led to accelerated grain filling, shortened effective grain-filling period, and reduced grain plumpness. Wen et al. (2000) found that droughts caused a decreasing area of sword leaves and reduction of chlorophylls, which in turn resulted in premature leaf senescence, lower grain weight, and reduced yields.
Many senescence-associated genes (SAGs) have been cloned (Quirino et al., 2000). Gan and Amasino et al. (1995) reported that specific expression of isopentyl transferase (IPI) gene, which is pivotally involved in the cytokinin synthesis pathway, significantly delayed senescence of the transgenic tobacco plants, and that tobacco plants containing the SAG12-IPT transgene had an increase of seed yield and dry weight by 50%. Yuan et al. (2002) found that after transformation of the SAG12-IPT fusion gene, the transgenic cabbages exhibited delayed senescence, increased harvest yields, and sustained freshness, offering a new vegetable breeding approach to increase the shelf life. Zhang et al. (2008) reported that the shotgun-transformed transgenic cool-season turfgrass tall fescue, which expressed the Leafy-ipt transgene, displayed significantly increased cold resistance and delayed senescence. Ding et al. (2007) found that rice transformed with the maize ppc transgene exhibited delayed senescence and significantly increased photosynthetic rate, especially under stress conditions.
Crop yield and quality are affected by a variety of environmental stresses, such as drought, flood, freezing, heat, and diseases. Among them, drought is one of the most prevalent and most frequent natural disasters, having the greatest negative impact on crop yields (Wang et al., 2007). Statistics from 1950 to 2007 showed that in China an average of 21,733,300 hm2 of agricultural lands had been stricken by drought every year, and an average annual loss of grain was up to 15.8 billion kilograms, which represented more than 60% of all grain losses caused by various natural disasters (Kuang et al., 2014). To alleviate the damages caused by drought, cultivating new drought-resistant crop varieties via plant genetic engineering has also become a powerful approach in addition to other integrated managements such as enhanced monitoring of environmental dynamics, improved risk assessment and improved water resource utilization.
Recent years many drought-related signal molecules, transcription factors and functional genes have been cloned, which include AREB1, AREB2, rd19, rd22, and transcription factors MYC, MYB, bZIP, etc. (Jiang et al., 2013; Umezawa et al., 2006). Taji et al. (2002) found that overexpression of galactinol synthase gene (AtGo1S2) in Arabidopsis increased the endogenous levels of galactinol and raffinose, reduced water evaporation in leaves and improved drought resistance. Haake et al. (2002) showed that transformation of Arabidopsis with the transcription factor CBF4 (which binds to CRT/DRE) transgene could result in enhanced activities of the TGGCCGAC element and its downstream genes, which in turn improved drought and cold tolerance in transgenic Arabidopsis. Pellegrineschi et al. (2002) transformed wheats with DREB1a, and found that under severe water stress, all control wheats withered and died while transgenic wheats survived. Capell et al. (1998) found that transforming rice with an oat arginine decarboxylase gene (Adc) could reduce loss of chlorophylls under drought stress and prevent transgenic rice from drought damage. Xu et al. (1996) transformed rice with barley HVA1 gene, which expresses a late embryogenesis abundant protein (LEA), and observed high expression levels of LEA in leaves of the transgenic rice, and found that both the drought and salt resistance of the second-generation transgenic rice were significantly improved. Under these stresses, the transgenic rice exhibited high growth rate, reduced stress-related symptoms, and faster post-stress recovery rate. Nelson et al. (2007) also demonstrated that transgenic maize overexpressing ZmNF-YB2, a homologous gene of the Arabidopsis transcription factor gene AtNF-YB2, had higher drought tolerance with significantly higher levels of chlorophylls, photosynthetic rate and stomatal conductance and significantly lower leaf temperature compared with control.
In summary, identification of genes related to delayed leaf senescence and increased stress tolerance together with subsequent manipulation of their expression by means of genetic engineering is an effective approach to obtain transgenic crops with delayed senescence and without increased growth cycle, which in turn may result in increased and sustained crop yields and improved crop quality, bringing about significant economic and social benefits.